Living cells respond to extracellular conditions through signaling cascades, which are mediated by a variety of protein modification reactions. One ubiquitous protein modification that regulates many metabolic and cell signaling pathways is kinase-catalyzed protein phosphorylation (FIG. 1A). Alteration of pathways involving kinases and phosphorylation can lead to diseases, such as Parkinson's, cancer and diabetes mellitus. Therefore, studying protein kinases and their phosphorylated substrates is critical to understand cell signaling pathways in both diseased and healthy cells.
With over 500 kinases and potentially thousands of phosphoproteins, multiple complementary approaches are necessary to monitor the complex cellular phosphoproteome. One powerful approach exploits analogs of the universal co-substrate of kinases, adenosine-5′-triphosphate (ATP, FIG. 1B). Multiple ATP analogs have been employed in kinase research, including base modified, sugar modified, and triphosphate modified analogs. Certain γ-phosphate modified ATP analogs have been used to label kinase substrates for subsequent purification and analysis. For example, ATP-biotin (1, FIG. 1B) is promiscuously accepted as a cosubstrate by protein kinases to phosphorylbiotinylate substrates. After kinase-catalyzed biotinylation with ATP-biotin, the biotin group facilitates analysis of phosphoproteins using various commercial streptavidin-conjugated reagents. Unfortunately, due to the impermeability of ATP analogs, ATP-biotin has been used in vitro only. The ability to utilize ATP-biotin in living cells would promote the study of protein kinases in more physiologically relevant conditions.
Accordingly, there is a need for cell-permeable ATP-biotin analogues.